The Development of Invertebrate Host Models for Burkholderia spp. Infection Studies

  • Zoe Freeman

Student thesis: Doctoral ThesisPhD

Abstract

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, an opportunistic but serious human disease endemic to Southeast Asia and Northern Australia. The ‘Bp-group’ includes Bp and the closely-related organisms B. thailandensis (Bt) and B. oklahomensis (Bo), all of which are usually soil-dwelling saprophytes, and B. mallei (Bm) which is an equine-host-adapted pathogen.Bt is virulent in a number of invertebrate models but is generally non-pathogenic for mammals and is often used as a surrogate for the study of virulence mechanisms shared with Bp. Experiments to assess the potential of the Tobacco Hawkmoth Manduca sexta as a model host for Bp or Bt infection revealed surprising results. Bp, Bt and Bo were all lethal to M. sexta larvae. This is the first report of Bo virulence in an infection model. Additionally, the relative virulence of the three species was the reverse of that reported in humans and in larvae of the Greater Waxworm Galleria mellonella. Despite that, well-known hallmarks of Bp-group pathogenesis in mammalian hosts – intracellular survival and multiplication, actin remodelling and acute sepsis – were observed in M. sexta infection during a fluorescent confocal microscopy time-course study.M. sexta feeding experiments with Bt and Bo indicated that cultures of these bacteria are also pathogenic via the oral route, which is likely to be relevant for natural insect-bacteria interactions. Cell-free supernatant of Bo was as harmful to larvae as complete culture, supporting previous suggestions that Bp-group bacteria produce toxins or paralytic agents that are active against invertebrates.Finally, Rapid Virulence Annotation (RVA) was performed as a genome-wide screen for virulence determinants of Bp strain K96423, using three invertebrate bioassays with a recombinant expression library. In response to problems with the reproducibility of biologically active clones, a new statistical approach was devised which enabled quantitative identification of the most convincing RVA hits.
Date of Award19 Jun 2013
LanguageEnglish
Awarding Institution
  • University of Bath
SupervisorNicholas Waterfield (Supervisor)

Keywords

  • RVA
  • rapid virulence annotation
  • thailandensis
  • invertebrate
  • manduca
  • sexta
  • larva
  • haemocyte
  • virulence factor

Cite this

The Development of Invertebrate Host Models for Burkholderia spp. Infection Studies
Freeman, Z. (Author). 19 Jun 2013

Student thesis: Doctoral ThesisPhD